The cap-binding complex modulates ABA-responsive transcript splicing during germination in barley (Hordeum vulgare)

To decipher the molecular bases governing seed germination, this study presents the pivotal role of the cap-binding complex (CBC), comprising CBP20 and CBP80, in modulating the inhibitory effects of abscisic acid (ABA) in barley. Using both single and double barley mutants in genes encoding the CBC, we revealed that the double mutant hvcbp20.ab/hvcbp80.b displays ABA insensitivity, in stark contrast to the hypersensitivity observed in single mutants during germination. Our comprehensive transcriptome and metabolome analysis not only identified significant alterations in gene expression and splicing patterns but also underscored the regulatory nexus among CBC, ABA, and brassinosteroid (BR) signaling pathways.


Global analysis of hvcbp20.ab/hvcbp80.b embryos transcriptome in response to ABA
To better understand how the double mutant responded to ABA during seed germination, we performed a comparative analysis of the transcriptomes of all genotypes in the presence of ABA and under control conditions.
Under control conditions, there were only a few transcriptomic changes in all tested mutants compared to the wild type (WT).However, ABA treatment significantly increased the numbers of DEGs and DETs (Table 1).The double mutant in CBP20 and CBP80 genes showed the greatest transcriptional changes in response to ABA compared to the other genotypes.The numbers of differentially alternatively spliced (DAS) genes, differentially expressed transcripts (DETs), and differential transcript usage (DTU) was the highest in the double mutant.This number in hvcbp20.ab,hvcbp80.b and WT were reduced by over 30% compared to the amount of DAS, DET, DTU identified in the double mutant.Moreover, the double mutation significantly affected alternative splicing and transcript isoform formation.The intersection between DE/DAS genes and DET/DTU showed that 69 genes underwent AS only in the WT, 60 in hvcbp20.ab,62 in hvcbp80.b,and 108 in the double mutant, corresponding to 68, 58, 60, and 71% of all DAS genes, respectively (Supplementary Fig. S2a).This suggests that DAS genes are mainly regulated at the alternative splicing level.Conversely, most DTU transcripts were produced by transcription in conjunction with AS, with DTU alone comprising 40% of the double mutant (Supplementary Fig. S2b).

Co-expression analysis reveals clusters of genes with a similar expression pattern in double mutant and WT in the presence of ABA
Co-expression analysis of DEGs (8070) identified in all genotypes tested in response to ABA during the seed germination revealed 12 gene clusters (Fig. 2a; Supplementary Data S1).The C8 and C11 clusters showed consistent expression patterns between the WT and the double mutant, contrasting with hvcbp20.ab and hvcbp80.b.In the C8 cluster, genes were upregulated in both the double mutant and WT, while being downregulated in the single mutants.On the contrary, the C11 cluster contained genes downregulated in the WT and double mutant but upregulated in hvcbp20.aband hvcbp80.bsingle mutants.The Gene Ontology (GO) enrichment analysis of these clusters identified key biological processes that were prevalent in DEGs with similar expression patterns.In the C8 cluster, the glycolipid catabolic process (GO:0019377), carbohydrate metabolic process (GO:0005975), maintenance of location (GO:0051235), vegetative phase change (GO:0010050) and glutamate catabolic process (GO:0,006538) were the most significant GO terms (Fig. 2b).Whereas in the C11 cluster the top GO-BP were amino acid transmembrane transport (GO:0003333), organic acid transmembrane transport (GO:1903825), protein palmitoylation (GO:0,018,345), negative regulation of floral organ abscission (GO:0060862) and negative regulation of dephosphorylation (GO:0035305) (Fig. 2c).

ABA-Induced expression of splicing factors and brassinosteroid signaling regulators unique to double mutant germination
We identified 642 DEGs specific to the double mutant.The single mutants hvcbp20.ab and hvcbp80.bdisplayed 241 and 591 unique DEGs, respectively, whereas the WT had 281 DEGs (Fig. 2d).Notably, the gene expression profile of the double mutant was significantly higher, with implications for the distinct from single mutants germination phenotype observed under ABA treatment.GO enrichment analysis of the upregulated gene set in the double mutant showed biological processes (BP) such as nucleic acid phosphodiester bond hydrolysis (GO:0090305)), suggesting an upregulation of splicing activity (Supplementary Data S2, Supplementary Data S3).Notably, six splicing factors have been identified, each with homologs in Arabidopsis, thereby providing a phylogenetic anchor for functional suggestions (Fig. 2e).These include CELL DIVISION CYCLE 5 (CDC5; BaR-T2v18chr5HG272090), U2af small subunit A (U2AF35A; BaRT2v18chr5HG254110), SUPPRESSOR OF ABI3-5 (SUA; BaRT2v18chr3HG159600), embryo-defective 2016 (EMB2016; BaRT2v18chr7HG366170), and putative branchpoint-bridging protein-like (SF1; BaRT2v18chr2HG081650) (Supplementary Data S4).Conversely, genes implicated in the negative regulation of brassinosteroid signaling pathways (GO:1900458) were downregulated, highlighting a potential mechanistic intersection between ABA and BRs signaling, REMORIN (REM4.1;BaRT2v18chr2HG069620), BRI1 KINASE INHIBITOR 1 (BKI1; BaRT2v18chr5HG251390), and a previously uncharacterized gene (BaRT2v18chr5HG272750) (Supplementary Data S5).Collectively, these results delineate a distinct transcriptional landscape for the double mutant, with implications for its exclusive ABA-mediated germination phenotype, mediated in part by modulations in the splicing and brassinosteroid signaling pathways.

Regulatory interplay between transcription factors, splicing factors, and brassinosteroid signaling inhibitors in double mutant
To elucidate the transcription factors (TFs) that potentially orchestrate the regulation of gene expression in response to ABA, we analyzed DEGs after ABA treatment across all genotypes (Supplementary Data S6).This yielded a list of 24 TFs in the double mutant, which starkly contrasts with the 6, 21, and 9 TFs found in hvcbp20.ab, hvcbp80.b,and WT, respectively.Notably, most TFs identified in the double mutant were upregulated, corroborating the enhanced transcriptional activity observed in these embryos upon ABA exposure.
The TFs encompassed 12 distinct transcription domain families (Supplementary Table 1), suggesting diverse regulatory capacities for modulating ABA responses during the seed germination stage.We next focused on prediction of transcription factor-binding sites (TFBSs) within promoters of DEGs specific to the double mutant.In response to ABA, six TFs were associated with 1178 TFBSs among the 392 DEGs specific to the double mutant suggesting potential regulatory complexity (Supplementary Data S7).A subset of these TFs was identified as having binding sites within DE genes encoding critical splicing factors.One TFBS was within the promoter of the negative regulator of BR signaling (REM4.1;BaRT2v18chr2HG069620) and one TFBS within serine/threonineprotein kinase SAPK10 (SAPK10; BaRT2v18chr7HG385230) (Supplementary Data S8).These results suggest that the identified TFs may modulate the expression levels of genes relevant to the seed germination phenotype of double mutants in the presence of ABA, which may be CBC-dependent.

Alternative splicing is impaired in germinating embryos of hvcbp20.ab/hvcbp80.b in the presence of ABA
To compare changes in the alternative splicing pattern in the double mutant, hvcbp20.ab,hvcbp80.b and the WT in response to ABA, the sets of DAS genes were compared (Fig. 3a).Most genes that underwent splicing were uniquely spliced in each genotype.However, the largest number of DAS genes was identified in the double mutant (Supplementary Data S9).To identify the specific transcripts contributing to the classification of a gene as DAS, we performed a differential transcript usage (DTU) analysis.This process identified individual transcripts that exhibited significant differences relative to the other transcripts from the same gene.In the double mutant, 72% of exclusive DAS genes contained DTU transcripts.For hvcbp20.ab and hvcbp80.b, the DAS&DTU amounted  www.nature.com/scientificreports/ to 64% and 66%, respectively.In contrast, the lowest number (58%) of exclusive DAS genes with corresponding exclusive DTU was identified in the WT (Fig. 3b; Supplementary Data S10).Hence, these data indicate that the

Transcripts of splicing factors accumulate during seed germination in double CBC mutant in the presence of ABA
To reveal significant differences in transcript abundance between the double mutant and the single hvcbp20.aband hvcbp80.bmutants, together with the WT in response to ABA, analyses were performed at the transcript level by examining the DET.The highest number of DETs (3404) was exclusively expressed in the double mutant.In the other genotypes, 1188 DETs were exclusive to hvcbp20.ab,2282 to hvcbp80.b and 1192 to the WT (Fig. 3c).As the CBC complex is involved in RNA metabolism, including AS, the analysis of isoforms in hvcbp20.ab/hvcbp80.bwill suggest the roles of the CBC in the response to ABA (Supplementary Fig. S3).The double mutant exhibited the highest number of events across most features of AS transcripts, particularly the premature termination codons (PTC) and non-sense mediated decay (NMD) categories, suggesting a possible interaction between the CBP20 and CBP80 genes in response to ABA treatment.GO enrichment analysis showed that genes encoding exclusively upregulated transcripts (DET) in double mutant are involved in GO 'biological process' (GO-BP) that involve metabolism and genes downregulated involve transcripts engaged in small molecule metabolic processes and anion and ion transport (Fig. 3d; Supplementary Data S11).Furthermore, to quantify the modifications in the AS machinery attributable to the double mutation, transcripts linked to RNA splicing (GO:0008380) were catalogued among the uniquely expressed DETs in the double mutant and compared to the single mutants and WT.This led to the identification of 61 genes that accounted for 72 unique DETs in the double mutant, a notable enrichment that may signify substantial reprogramming of the splicing landscape, indicative of an adaptive response to ABA (Fig. 3e; Supplementary Data S12).

Predicted interactions of CBC complex subunits with RNA metabolism and splicing factors
To test whether the proteins encoded by the DEGs and DAS genes, identified specifically for hvcbp20.ab/hvcbp80.bembryos in the presence of ABA, demonstrated direct physical interactions with the CBC subunits, a putative model of protein-protein interaction (PPi) was devised using the STRING database (Fig. 4, Supplementary Data S13).Utilizing CBP20 and CBP80 as seeds in this analysis, our search revealed 28 proteins that exhibited physical interactions with CBC subunits, as corroborated by the experimental data deposited in STRINGdb.A breakdown of these interactions revealed that 20 proteins interacted with both CBP20 and CBP80, while four interacted only with the CBP20 subunit, and another four with CBP80 (Supplementary Data S14).Intriguingly, within this interactome, seven genes were classified as DEGs and two as DAS, identified specifically in hvcbp20.ab/hvcbp80.b.These genes predominantly function in RNA processing and splicing, thereby highlighting the interconnectedness between these processes and the CBC complex.Notably, these DEGs are genes encoding SF, including arginine/serine-rich 4 (BaRT2v18chr1HG011980), which interacts with both CBC subunits.Moreover, two splicing factors that interact solely with CBP20 are noteworthy: the splicing factor U2af small subunit A (U2AF35A; BaRT2v18chr5HG254110) and the CELL DIVISION CYCLE 5-like protein (CDC5; BaRT2v18chr5HG272090) as detailed in Supplementary Data S14.These results showed an association between the CBC complex and other splicing and RNA processing factors, confirming its involvement in the regulation of seed germination via AS.The proteins encoded by these genes might act as CBC-dependent regulators of seed germination in the presence of ABA.

Brassinosteroid biosynthesis may be increased in hvcbp20.ab/hvcbp80.b mutant in the presence of ABA
Alterations in BR signaling were observed in the double mutant through transcriptome profiling.Gene ontology enrichment analysis of genes specifically upregulated in double mutant demonstrated that the 'negative regulation of BR signaling' was overrepresented as the top Biological Process of GO categories.Additionally, genes encoding negative regulators of BR signaling were exclusively downregulated in the double mutant in response to ABA.To determine whether the ABA-insensitive phenotype also involves changes in BR biosynthesis, we analyzed distinctive the metabolomic profiles of germinating embryos from all genotypes under ABA treatment (Fig. 5a).The metabolomes were interrogated, and the sources variation were identified.Focusing on those sources of variation linked to hormones, this revealed significantly increased levels of 6-alpha-hydroxy-6-deoxycastasterone, a brassinolide biosynthesis intermediate, in the double mutant when compared to the WT and other genotypes.This upregulation aligns with the observed germination patterns, in which the double mutant demonstrated an enhanced germination rate, suggesting a growth-promoting effect and diminished ABA sensitivity (Fig. 5b).

Towards Sebastian RTD: capturing genotype-specific transcriptome variations
To assess whether the BaRTv2.18transcriptome 51 , based on the Barke genotype, potentially overlooked transcriptomic data specific to the Sebastian genotype, adhered to the BaRTv2.18assembly process the Reference Transcriptomic Dataset (RTD) named BarkeRTD was generated.This included assembling reference transcript datasets (RTDs) from Illumina RNA-seq short reads (RNAseq RTD) and PacBio Iso-seq long reads (Isoseq RTD comprised eight samples from this study representing each genotype under control and ABA), by using the same Barke reference genome as outlined by Ref. 51 .RNAseq RTD and Isoseq RTD were merged to produce a unified BarkeRTD.Comparative analysis indicated that BaRTv2.18exhibits slightly higher genome coverage than Bark-eRTD.This was due to the mapping of the Sebastian sequencing data to the Barke reference genome, potentially leading to the omission of Sebastian-specific genes 52 .However, BarkeRTD demonstrated notable augmentation, featuring approximately 5000 additional genes and 9000 more transcripts (Supplementary Table 2).www.nature.com/scientificreports/ The integration of RNAseq and Isoseq RTDs resulted in a substantial contribution, introducing over 12,000 novel splice junctions and 25,700 novel intron combinations to enrich the BarkeRTD, surpassing the content available in BaRTv2.18 (Fig. 6a,b).It is worth to note that none of the tissues used to produce BaRTv2.18represent germinating barley embryos and we therefore prepared an RTD, also against Barke, that utilised our sequence data to provide sequence information that may be additional to BaRTv2.18.Our commitment to enhancing the dataset continues with the ongoing generation of new Iso-seq data.We are optimistic about achieving an even more comprehensive and refined Sebastian RTD using a Sebastian-based reference genome, coupled with additional sequencing data in the future.

Discussion
Mutations in CBP20 and CBP80 confer hypersensitivity to ABA during seed germination in Arabidopsis and barley (Hordeum vulgare) [43][44][45][46][47][48] .Therefore, we expected the same ABA phenotype for the hvcbp20.ab/hvcbp80.bdouble mutant.However, our results demonstrated that the germination rate of the double mutant was comparable to that of the wild type (WT) in the presence of ABA, which is in striking contrast to the inhibition of germination in single mutants.
In Arabidopsis, the cos1 coi1-2 double mutant exhibits resistance to pathogens and a deviation in senescence patterns 53 , mirroring the phenomenon described in this study in the double mutant hvcbp20.ab/hvcbp80.b.A reversal of the effect of the mutation in the da3-1 single mutant, which leads to curly leaves and increased organ sizes, was observed in sud1-1 da3-1 and sud2-1 da3-1 double mutants 54 .Similarly, the growth of bri1-5/ bri1-1D, bri1-5/brs1-1D and bri1-5/bak1-1D double mutants is partially restored compared to the dwarf bri1-5 single mutant with the mutation in the BRASSINOSTEROID-INSENSITIVE 1 (BRI1) gene from the BR signal transduction pathway 55 .
In all these examples, the double mutants complemented the genetic relationship, presenting a reversion to a WT-like phenotype that was not observed in the single mutants.This could imply a buffering effect or compensatory genetic interaction, where the confluence of two mutations mitigates the sensitivity observed in the single mutants.We cannot rule out a scenario in which the two subunits of the CBC can interact when both are mutated, while the interaction between mutated CBP20 and WT CBP80 or vice versa is not efficient.However, further experimental validation is required to confirm this.The double mutant insensitivity to ABA also suggests compensatory transcriptional reprogramming, possibly through altered expression or function of key genes within the ABA response network.This hypothesis was supported by the elevated transcriptional activity of the double mutant.Comparative transcriptome analysis revealed that the double mutant exhibited the most significant changes after ABA treatment, with a 30% increase in differentially expressed genes and transcripts relative to other genotypes.The prominence of differentially alternatively spliced genes in the double mutant suggests a reconfiguration of the splicing landscape, potentially contributing to the observed phenotype.The action of the CBC complex is notably more important in response to stress or ABA, evidenced by the minimal transcriptomic changes observed in the studied mutants compared to the WT under control conditions.These findings align with previous studies on the CBC complex in barley and Arabidopsis under stress conditions 43,56 .
In plants, the involvement of the CBC complex in the constitutive and alternative splicing of pre-mRNA has been documented, where the CBC, together with the zinc finger protein SERRATE (SE), acts as a platform for interaction with other splicing factors 37,38 .Thus, we hypothesized that the role of CBC in seed germination may be splice-dependent.RNA-seq analysis of the embryo transcriptome in response to ABA revealed that genes and transcripts encoding splicing factors were specifically downregulated in the double mutant compared to hvcbp20.ab, hvcbp80.b,and WT.Among the DEG, we identified six genes encoding splicing factors that were upregulated only in the double mutants in the presence of ABA.One of them was CELL DIVISION CYCLE 5 (CDC5) which encodes a Myb-related protein that is a subunit of the nuclear MOS4-ASSOCIATED COMPLEX (MAC) in Arabidopsis, important for the regulation of plant innate immunity 57 .Interestingly, CDC5 interacts with SE www.nature.com/scientificreports/and DICER-LIKE 1 (DLC1) during miRNA biogenesis and mediates alternative splicing of RESISTANCE TO RALSTONIA SOLANACEARUM 1 (RRS1) and RPS4 [58][59][60] .The interactome analysis revealed a physical interaction between CBP20 and CDC5.The next step is to validate the relationship between the CBC complex, CDC5, and SE in AS regulation during seed germination.The other identified splicing factor is the U2 auxiliary factor (U2AF), which is a part of the core of the spliceosome 61 .U2AF is a heterodimer composed of the U2AF35 and U2AF65 subunits.U2AF35 binds to the 3′ splice site, whereas U2AF65 binds to the polypyrimidine tract (PPT) of nascent pre-mRNA 62 .Moreover, U2AF35 undergoes alternative splicing to form two isoforms, U2AF35a and U2AF35b, which interact with U2AF65 63 .Other studies in Arabidopsis have shown that U2AF65a and U2AF65b interact with SPLICING FACTOR 1 (SF1) 64,65 .SF1 acts as a splicing enhancer and is a member of the serinearginine-rich (SR) protein family that facilitates spliceosome assembly 64,[66][67][68][69] .Several studies have revealed that SR proteins regulate plant responses to environmental stress via AS [70][71][72][73] .
Our study suggests a physical interaction between CBP20 and U2AF35a (BaRT2v18chr5HG254110) based on interactome analysis and identified increased expression of the putative branchpoint-bridging protein-like (SF1; BaRT2v18chr2HG081650), suggesting its involvement in the regulation of seed germination.Another factor from the SR family identified in our study that probably interacts with both CBC subunits is an arginine/serine-rich 4 (BaRT2v18chr1HG011980) a homolog of AtRS2Z33.In Arabidopsis, ectopic expression of AtRS2Z33 affects the AS of other genes, as well as the AS of its pre-mRNAs, generating splice variants containing premature termination codons (PTC), which are then directed into the nonsense-mediated decay (NMD) pathway.It ensures an important post-transcriptional mechanism linking AS with NMD and regulates cellular protein levels in the cells 74,75 .Taken together, the altered expression of these splicing factors exclusively in hvcbp20.ab/hvcbp80.bsuggests their potential dependency on the entire CBC complex.In addition, the potential interaction between the subunits of the CBC complex and the homologs of AtCDC5, AtU2AF35b/AtU2AF35a, and AtRS2Z33 suggests their common role during germination in the presence of ABA, which should be further investigated.We speculate that the 1.5-fold higher amount of DAS in hvcbp20.ab/hvcbp80.bcompared to hvcbp20.ab,hvcbp80.b,and WT is related to the altered expression patterns of the identified SFs.
Raczynska et al. 38 investigated young Arabidopsis seedlings and suggested that the entire CBC complex is involved in alternative splicing, with an emphasis on the first intron of the transcript.The CBP80 subunit plays a substantial role in alternative splicing, as demonstrated by the fact that the Atcbp80/abh1 mutant and the double mutant displayed significantly more common changes in alternative splicing levels than the Atcbp20 mutant and the double mutant.In our study, we did not observe such a relationship because there were 17 common DAS genes for hvcbp20.aband the double mutant and 10 for hvcbp80.b and the double mutant.Most DAS genes (94 DAS) were exclusive to hvcbp20.ab/hvcbp80.b, of which 76 DAS (81%) contained DTU transcripts.This indicated the importance of the entire barley CBC complex interaction in AS regulation during the seed germination stage.
Our approach, focusing on gene and transcript level expression changes, aimed to comprehensively investigate how the CBC influences alternative splicing dynamics, resulting in the generation of diverse transcript isoforms.This allowed us to observe the effect of CBC on AS not only at the gene level but also directly at the transcript level.Mutations in both CBC subunits led to a significantly increased accumulation of SF transcripts in the hvcbp20.ab/hvcbp80.bmutant.Among the 72 identified DET in SF, the presence of isoforms encoding homologs of AtSE, AtPRP4KA, AtPININ, and AtSR45 were particularly interesting.We observed the accumulation of two SF-coding transcripts (BaRT2v18chr7HG374470.5, BaRT2v18chr7HG374470.3), specifically in hvcbp20.ab/hvcbp80.b.Recent studies have shown that SE is also regulated by phosphorylation by the pre-mRNA PROCESSING FACTOR 4 KINASE A (PRP4KA), affecting its protein activity and proper accumulation in cells 76 .The PRP4KA transcript (BaRT2v18chr5HG220870.3) was also strongly upregulated in hvcbp20.ab/hvcbp80.bembryos in the presence of ABA.These results suggest a potential relationship among CBC, SE, and PRP4KA in fine-tuning germination in response to ABA, which requires further evaluation.We detected increased expression of two transcripts of the AtSR45 homolog (BaRT2v18chr3HG170170.2 and BaRT2v18chr3HG170170.8), which is a highly conserved RNA-binding protein.SR45 interacts with the ABA-dependent splicing factors SNW/ SKI-INTERACTING PROTEIN (SKIP) and SUA, and approximately 30% of the transcripts that bind to SR45 are involved in the ABA-signaling pathway 77,78 .Moreover, SR45 interacts with other SFs, including U2AF35b and its paralogs U2AF35, RSZ21, and SR34a, which were upregulated in our study (BaRT2v18chr3HG143420.7,BaRT2v18chr6HG321740.4, BaRT2v18chr6HG321740.5, and BaRT2v18chr2HG059400.6) [79][80][81][82] .This suggests the existence of an ABA-dependent interaction network that facilitates spliceosome assembly at the seed germination stage, in which the CBC complex is involved.
The upregulation of specific transcription factors in the double mutant further suggests a broad regulatory capacity, with the potential to reshape the ABA response during the germination stage.The highest number of TFs identified in hvcbp20.ab/hvcbp80.bcorresponded to the largest changes in DEG and DET levels in this mutant after ABA treatment.Five of the exclusively expressed TFs were particularly interesting because they potentially bind to the promoters of the splicing factors AtCDC5, AtEMB2016, AtSF1, AtRS2Z33, AtU2AF35b/AtU2AF35a and AtSUA.These TFs are likely to also regulate AtREM4.1 and the SAPK10 homolog of AtSnRK2s is important for ABA and BRs signaling.
ABA and BR antagonistically regulate various plant developmental processes, including seed germination 23,83 .A particularly interesting finding was that the 'negative regulation of brassinosteroids' was within the most significant biological process (BP) in the GO category in germinating embryos of double mutant in the presence of ABA.Interestingly, negative regulators of BR signaling were specifically downregulated in the double mutant (Supplementary Data S5).We detected reduced expression of REMORIN (REM4.1;BaRT2v18chr2HG069620) and BRI1 KINASE INHIBITOR 1 (BKI1; BaRT2v18chr 5HG251390).In Arabidopsis, insertional mutants in REM4.1 exhibit strong insensitivity to ABA during seed germination 84 .This mutant was even more insensitive than the abi1-1 (abscisic acid-insensitive 1-1) mutant 85 .When the level of ABA in the cell increases, OsREM4.1 is repressed by OsbZIP23 (OsABF4).Subsequently, OsREM4.1 inhibits BR signaling by disrupting the complex formation of OsBRI1 and OsSERK1 86 .In our study, we did not identify a homolog of the bZIP23 transcription factor among exclusive DEGs in hvcbp20.ab/hvcbp80.b.However, we observed reduced expression of the transcription factor TGACG MOTIF-BINDING FACTOR 6 (TGA6; BaRT2v18chr1HG033440), which belongs to the basic leucine zipper (bZIP) gene family.TGA6 contains a TFBS in the promoter sequence of REM4.1, thus potentially regulates REM4.1.
In addition, among the specifically downregulated genes in the presence of ABA in hvcbp20.ab/hvcbp80.b,we observed the gene encoding the serine/threonine-protein kinase SAPK10 (SAPK10; BaRT2v18chr7HG385230), which is highly homologous to SnRK2s in Arabidopsis.ABA induces OsSAPK10 and its overexpression confers hypersensitivity to ABA 91 .In plants, SnRK2 kinases physically bind and phosphorylate ABF to transmit ABA signals 92 .Moreover, our interactome analyses showed that upregulated transcription of TF from the CYSTEINE-RICH POLYCOMB-LIKE PROTEIN (CPP) family (BaRT2v18chr1HG048310) that potentially binds to SAPK10.To understand the regulatory mechanism, we need to confirm whether the transcription factor CPP interacts with SAPK10 in planta and whether SAPK10 adds a phosphate group to TGA6, further influencing REM4.1.Additionally, the metabolome results showed increased concentrations of castasterone, which is a precursor to brassinolide (BL), the most active BR 93,94 , in germinating embryos of hvcbp20.ab/hvcbp80.band WT plants in the presence of ABA.Considering these results, we speculate that the downregulation of REM4.1 and BKI1 genes from the BR signaling pathway in the presence of ABA, together with potentially increased BR biosynthesis, resulted in the promotion of seed germination in the double hvcbp20.ab/hvcbp80.bmutant.Our findings demonstrate that the disruption of both CBC subunits potentially impairs the crosstalk between ABA and BR signaling and may induce BR biosynthesis.However, further research is required to understand the biological and genetic bases of these observations.We propose a hypothetical model of action for the identified factors in hvcbp20.ab/hvcbp80.bexplaining its seed germination phenotype in the presence of ABA linked to signaling (Fig. 7).
In conclusion, our investigation demonstrated that the barley double mutant hvcbp20.ab/hvcbp80.bexhibits resistance to ABA during seed germination, suggesting that simultaneous mutations in the CBC subunits can mitigate ABA sensitivity through genetic interactions.Our detailed transcriptomic analysis of ABA responses highlights significant transcriptional reorganization, marked by shifts in gene expression, alternative splicing, and transcript isoform variation.The distinct phenotypes of the double mutant likely arise from the activation of specific gene and transcript sets depending on both CBC subunits.This study is the first to establish the crucial role of CBC in ABA-mediated seed germination in barley, particularly through its influence on alternative splicing and its interplay with the ABA and BR signaling pathways.Moreover, we have identified several key components of signaling pathways dependent on CBC, and thus warrant further investigation, which paves the way for future studies to comprehensively decode CBC function in ABA signaling during germination.

Plant material
We used grains and embryos of barley TILLING mutants: the hvcbp20.ab, in the CBP20 gene (CAP-BINDING PROTEIN 20; BaRT2v18chr6HG306340; Refs. 43,95), and hvcbp80.b, in the CBP80 gene (CAP-BINDING PROTEIN 80; BaRT2v18chr4HG195950), single mutants, the hvcbp20.ab/hvcbp80.bdouble mutant, and the 'Sebastian' wild-type (WT), all sourced from the HorTILLUS population 96 .Single mutants were identified through TILL-ING in the M 2 generation (then backcrossed with WT to clean the genetic background), and the double mutant was identified via genotyping the F 2 progeny following crossbreeding of the single mutants (Supplementary Fig. S1).Mutations in the CBP20 and CBP80 genes in hvcbp20.ab/hvcbp80.bdouble mutant were confirmed using Sanger sequencing in three biological replicates in two consecutive generations of double mutant after self-pollination series.

Seed germination assay in the presence of 75 µM ABA, embryos isolation and RNA extraction
Seeds were sterilized in 20% sodium hypochlorite solution for 20 min and washed with sterilized water for 5 min.They were then placed in Petri dishes with three layers of filters and treated with either sterilized water or sterilized water containing 75 μM ABA (cis-trans-abscisic acid; catalog no.862169; Sigma-Aldrich).The seeds were stratified for 4 d at 4 °C and then transferred to a growth chamber.Germination was defined as the visible emergence of the radicle through the seed coat and was assessed on 1 and 7 DAI (days after imbibition).The assay was performed in three biological replicates, each comprising 30 seeds of each genotype per petri dish.At 1 DAI, the embryos were isolated from the endosperm using a sharp scalpel blade, placed in microcentrifuge tubes (Eppendorf) containing RNAlater reagent, and stored at 4 °C until RNA isolation.RNA from isolated germinating embryos at 1 DAI was extracted using the mirVana™ Isolation Kit (Ambion, USA) following the manufacturer's instructions.RNA was isolated in four biological replicates, each consisting of 20 embryos.In total, 32 RNA samples were extracted.RNA concentration and quality were assessed using a NanoDrop spectrophotometer (NanoDrop Technologies, Wilmington, USA) and Agilent Bioanalyzer (Agilent Technologies, Santa Clara, USA).

Library construction and Illumina sequencing
In total, 32 RNA samples (30 µL each) obtained from the germinating embryos at 1 DAI of four tested barley genotypes ('Sebastian' (wild-type), hvcbp20.ab,hvcbp80.b,hvcbp20.ab/hvcbp80.b),each in four biological replicates, subjected to two treatment conditions (control and 75 µM ABA) at 1DAI, were used for TruSeq stranded mRNA cDNA library construction and next-generation sequencing (NGS) (Macrogen Inc., South Korea).Illumina RNA-seq was performed using a NovaSeq6000 sequencer ( reads was performed using FastQC, adapters were removed with Cutadapt, and quality control of the trimmed reads was checked again with FastQC 97 .Low-quality reads were filtered using Cutadapt software.Paired-end reads were mapped to the BaRTv2.18 51barley reference transcriptome using the Kallisto tool 98 .The mapped reads were quantified and normalized to transcripts per million (TPM) using Kallisto.

Differential expression analysis
Differential gene and transcript expression analyses were performed using the limma-voom package in a 3D RNA-seq application 99 .Analysis was performed for two treatment groups (ABA and control) and seven comparisons (contrast groups): under control conditions between the hvcbp20.ab,hvcbp80.b,hvcbp20.ab/hvcbp80.bmutants against the 'Sebastian' parental variety (control.cbp20-control.WT, control.cbp80-control.WT, control.double-control.WT) and after ABA treatment of each genotype (hvcbp20.ab,hvcbp80.b,hvcbp20.ab/hvcbp80.band wild type 'Sebastian') against control conditions (ABA.WT-control.WT, ABA.cbp20-control.cbp20,ABA.cbp80-control.cbp80,ABA.double-control.double).In all contrast groups, differentially expressed genes (DEG), which show significant changes in expression levels between tested conditions; differentially expressed transcripts (DET), specific transcripts of genes exhibiting significant expression changes compared to other transcripts www.nature.com/scientificreports/within the same gene; differential alternative splicing (DAS), where genes with multiple transcript isoforms show varying abundance patterns between conditions; and differential transcript usage (DTU), highlighting specific transcript variants preferentially expressed between tested conditions, were analyzed.The threshold for significant differential gene and transcript expression was log 2 FC ≥ 1.5 or ≤ -1.5, with a Benjamini-Hochberg (BH) adjusted p-value < 0.01.To identify significant DAS, the percentage spliced (ΔPS) threshold was set at 0.5.The DAS genes within each contrast group were identified based on an adjusted p-value below the predefined threshold (< 0.01) and the presence of at least one transcript exhibiting a ΔPS greater than 0.5.For DTU identification, each transcript's expression was compared to the weighted average expression of all other transcripts within the same gene.
A transcript was classified as DTU if its adjusted p-value was below the predefined threshold (< 0.01) and the absolute value of ΔPS exceeded the defined cut-off value of 0.5.To identify common DEGs and DETs between all tested genotypes, and exclusive for each genotype in the treatment groups, Venn diagrams were constructed using the Venny 2.1 tool (https:// bioin fogp.cnb.csic.es/ tools/ venny/).In this study, we focused on the genes and transcripts specific for hvcbp20.ab/hvcbp80.b.Gene Ontology enrichment analysis was performed using TopGO (version 2.50.0)implemented in R (version 4.2.1) with an adjusted p-value < 0.01.The BH method was used to correct the False Discovery Rate (FDR).

Transcription factors (TFs) identification and binding site prediction
The PlantRegMap tool was used to predict transcription factors (TFs) 100 .Promoter sequences, defined as the 1500 bp upstream flanking regions, were retrieved through EnsemblPlants v. 45 using the BioMart tool, based on the MorexV3 barley genome version integrated into the EnsemblPlants database 101 .These promoter sequences were analyzed for transcription factor-binding sites (TFBS) using the PlantRegMap Binding Site Prediction feature.Subsequently, datasets detailing TFs and their potential target genes were merged to explore the potential regulatory interactions.

Identification of Arabidopsis homologs
The BLAST tool within the Ensembl Plants database was used to identify Arabidopsis homologs (version of the genome TAIR10).A threshold of at least 40% similarity between sequences was set for homology 102 .

Analysis of interactome
Using the STRING database (version 11.5), a predicted network of physical protein-protein interactions (PPIs) was constructed for the DEGs and DAS genes in the hvcbp20.ab/hvcbp80.bmutant in ABA presence.For network analysis, the physical subnetwork was selected, and the default parameters were used with a medium confidence interaction score threshold of 0.4 and no additional interactors.The PPi network was visualized using Cytoscape software 103 .

Co-expressed genes analysis
All significantly differentially expressed genes (DEGs) identified within the RNA-seq experiment of germinating embryos at 1 DAI from tested mutants and WT were used to identify gene co-expression clusters.The average transcript per million (TPM) gene values of four biological replicates of the ABA-treated samples were used in the ' clust 1.10.8'tool in Linux with default settings was employed for this purpose.Heatmaps were generated using the R package 'Pheatmap' v.1.0.12, based on TPM values.

Prediction of splicing factors
Splicing factors (SF) were searched by filtering the gene ontology (GO) term 'RNA splicing' (GO:0008380) from a list of exclusively differentially expressed genes (DEG) and a list of exclusively differentially expressed transcripts (DET) in each genotype studied under ABA vs. control conditions.

Metabolome analysis
Embryos extracted from germinating seeds were homogenized in liquid nitrogen and subsequently chilled on ice.Precise 40 ± 1 mg of this tissue was then transferred into microcentrifuge tubes, followed by the addition of 1 mL of chloroform: methanol: dH 2 O mixture (1:2.5:1).The samples were vigorously mixed using by vortexing at 4 °C for 15 min before being placed back on ice.Afterward, they were centrifuged at 4 °C at 5000×g for 3 min.The supernatants, which contained both polar and nonpolar metabolites, were carefully transferred to new tubes and dried using a Buchi Rotavapor system at 25 °C, to avoid complete evaporation.Finally, 100 µL of the supernatant was reserved for metabolomic profiling using liquid chromatography-tandem mass spectrometry (LC-MS/MS) as described by Baptista et al. 104 .

PacBio sequencing and reference transcriptome dataset (RTD)
Eight RNA SMRTbell Iso-seq libraries were prepared using RNA extracted from the same samples as those used for Illumina sequencing but pooled for each genotype.Further libraries were sequenced using the PacBio sequel II platform according to the manufacturer's protocol (Macrogen Inc., Seoul, Republic of Korea).To investigate whether transcriptomic analysis specific to the Sebastian genotype was overlooked using BaRTv2.18,a Barkebased transcriptome, and assess the potential necessity of assembling a Sebastian-specific reference transcript dataset (RTD) in future endeavors, we adhered to the BaRTv2.18assembly pipelines, assembling RTDs from both Illumina RNA-seq short reads and PacBio Iso-seq long reads 51 .Specifically, the STAR mapping tool 105 was used to align the trimmed RNA-seq reads of the 32 samples to the Barke reference genome 106 .Transcript models were assembled from the read alignment using two assemblers: Stringtie 107 and Scallop 108 .The resultant transcripts in

Figure 2 .
Figure 2. Global analysis of differentially expressed genes (DEG) of hvcbp20.ab,hvcbp80.b,hvcbp20.ab/hvcbp80.b and WT after 75 µM ABA treatment compared to control conditions.(a) Co-expressed clusters of DEGs in response to ABA.(b) Heatmap of DEGs and enriched GO biological processes in the C8 cluster.(c) Heatmap of DEGs and enriched GO biological processes in the C11 cluster.(d) Venn diagram of DEGs displaying up-and downregulated exclusively expressed genes in each contrast group.(e) Overrepresented GO biological processes among upregulated and downregulated exclusive DEGs in hvcbp20.ab/hvcbp80.b.

Figure 3 .
Figure 3. Global analysis of genes with differential alternative splicing (DAS) genes and differentially expressed transcripts (DET) in hvcbp20.ab,hvcbp80.b,hvcbp20.ab/hvcbp80.band WT after 75 µM ABA treatment compared to control conditions.(a) Venn diagram of DAS genes in each contrast group.(b) Venn diagrams displaying the amount of exclusive DAS genes with DTU transcripts.(c) Venn diagram of DETs displaying up-and downregulated exclusively expressed transcripts in each genotype.(d) Overrepresented GO biological processes among upregulated and downregulated exclusive DETs in hvcbp20.ab/hvcbp80.b.(e) The number of genes encoding transcripts of splicing factors (SF) with the corresponding number of transcripts of SFs among exclusive DET in each genotype.

Figure 6 .
Figure 6.Comparisons of BarkeRTD, Isoseq RTD, RNAseq RTD and BaRTv2.18 on (a) splice junctions and (b) intron combinations of multi-exon transcripts.Splice junctions or Intron combinations shared by multiple transcripts were only counted once.

Figure 7 .
Figure 7. Hypothetical model explaining the seed germination phenotype of the hvcbp20.ab/hvcbp80.b in the presence of ABA linked with BR signaling.In the presence of ABA in the hvcbp20.ab/hvcbp80.bdouble mutant, the unknown transcription factor from the CPP-family is exclusively upregulated.This transcription factor potentially binds SAPK10 and inhibits its expression.It leads to reduced phosphorylation of the TGA6 transcription factor by SAPK10 and reduced expression of the REM4.1 gene encoding a negative regulator of BR signaling.Additionally, in hvcbp20.ab/hvcbp80.b, in response to ABA, the BKI1 gene encoding the negative regulator of the BR signaling pathway is exclusively downregulated.All this together causes the seed germination process in the hvcbp20.ab/hvcbp80.bdouble mutant to be inhibited in the presence of ABA.The dashed lines represent possible interactions.The solid lines represent known interactions.The green color indicates exclusively differentially upregulated genes in hvcbp20.ab/hvcbp80.b in response to ABA.The red indicates exclusively differentially downregulated genes in hvcbp20.ab/hvcbp80.b in response to ABA.Pphosphorylation.Illustration created with BioRender.

Table 1 .
Number of DE/DAS genes and DE/DTU transcripts in different contrast groups.DE differential expression, DAS differential alternative splicing, DTU differentia transcript usage.

DE genes DAS genes DE transcripts DTU transcripts
40 M, 2xPE 150 bp).Preprocessing of the raw